JOURNAL OF BACTERIOLOGY, Apr. 1975, p. 206-214 Copyright 0 1975 American Society for Microbiology
Vol. 122, No. 1
Printed in U.S.A.
Maltose Chemoreceptor of Escherichia coli GERALD L. HAZELBAUER The Wallenberg Laboratory, University of Uppsala, Uppsala, Sweden Received for publication 15 November 1974
Strains carrying mutations in the maltose system of Escherichia coli were assayed for maltose taxis, maltose uptake at 1 and 10 qM maltose, and maltose-binding activity released by osmotic shock. An earlier conclusion that the metabolism of maltose is not necessary for chemoreception is extended to include the functioning of maltodextrin phosphorylase, the product of malP, and the genetic control of the maltose receptor by the product of malT is confirmed. Mutants in malF and malK are defective in maltose transport at low concentrations as well as high concentrations, as previously shown, but are essentially normal in maltose taxis. The product of malE has been previously shown to be the maltose-binding protein and was implicated in maltose transport. Most malE mutants are defective in maltose taxis, and all those tested are defective in maltose transport at low concentrations. Thus, as previously suggested, the maltosebinding protein probably serves as the recognition component of the maltose receptor, as well as a component of the transport system. Some malE mutants release maltose-binding activity and are tactic toward maltose, although defective in maltose transport, implying that the binding protein has separate sites for interaction with the chemotaxis and transport systems. Some mutations in lamB, whose product is the receptor for the bacteriophage lamba, cause defects in maltose taxis, indicating some involvement of that product in maltose reception.
The phenomenon of chemotaxis by Escherichia coli appears to result from the effect that a temporal gradient of an active compound has on the direction of rotation of bacterial flagella (23). Counter-clockwise rotation of flagella produces smooth swimming, and clockwise rotation produces a "tumble" (8, 9, 35) which results in a change of direction when swimming is resumed (10). An increase in attractant concentration shifts the balance between these two modes to smooth swimming (10, 13, 25), whereas a drastic decrease shifts it to tumbling (25). The effect of an increase or decrease in repellent concentration is the opposite from that of an attractant (23, 37, 38). Thus, bacteria tend to move up a spatial gradient of attractant and down one of repellent. Changes in concentration of chemotactically active compounds are detected by specific "chemoreceptors." Adler demonstrated that the functioning of these chemoreceptors does not require metabolism or general transport of the compound, but only requires the recognition of the molecule, presumably by a specific binding (1). In the simplest case, a given chemoreceptor includes all those structures involved in the
detection of one class of compounds and not in the detection of other classes. (There is genetic evidence that some chemoreceptors "overlap" [29; J. Adler, personal communication].) Receptors are linked to a "common pathway" through which recognition is translated into an effect on the flagella (1). Mutants are available that are defective in a particular chemoreceptor (4, 17, 26, 28, 29, 38). Such mutants, defective in the galactose receptor, made possible the identification of the galactose-binding protein (6) as the recognition component of that receptor (16). Boos has shown that this protein is a part of the 3-methylgalactoside transport system (11), which transports low concentrations of galactose into the cell (21, 33). The galactose-binding protein is released from the envelope of E. coli by osmotic shock, i.e., it is a periplasmic protein (18). A search for other shock-released binding proteins that might serve as chemoreceptor recognition components produced a ribose-binding activity and a maltose-binding activity (16). Later, a ribosebinding protein which probably functions in a ribose receptor was purified from Salmonella typhimurium (5). The maltose-binding activity
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MALTOSE CHEMORECEPTOR OF E. COLI
was purified as a maltose-binding protein and shown to be the product of the gene malE by Kellermann and Szmelcman (22). Recognition components for the glucose and mannose receptors have been identified as enzymes II of the phosphotransferase transport system by the characterization of the chemotactic behavior of enzyme II mutants and preliminary results indicate that the enzymes II for D-fructose and D-mannitol serve as the receptors for those two sugars, respectively (3). Genetic studies indicate that the galactose receptor has a second component in addition to the binding protein (28). These biochemical and genetic identifications constitute the information presently available about bacterial chemoreceptors. This paper is a beginning of the characterization of the maltose receptor of E. coli. Here the involvement in taxis toward maltose of the gene products of the known maltose operons is determined. Mutations that specifically affect the metabolism of maltose (thus excluding mutations in the catabolite repression system and the phosphotransferase system) are grouped in the two regions on the genetic map of E. coli (34). The malA region at 65 min contains malT, which codes for the positive regulatory molecule controlling all three maltose operons, and an operon consisting of malP, which codes for maltodextrin phosphorylase, and malQ, which codes for amylomaltase (34). The malB region at 79 min (Fig. 1) contains two divergent operons which have adjoining, and perhaps overlapping, promotor regions (19, 20). Mutants in malE, malF, or malK are defective in maltose transport and thus are unable to grow on maltose as a carbon source. The maltose-binding protein is the product of malE (22). The product of lamB is a protein that serves as the outer membrane receptor for the bacteriophage lambda (31). This protein is apparently also involved in uptake of low concentrations of maltose (M. Hofnung, personal communication). The involvement of the lambda receptor in maltose chemoreception will be discussed Leftward poLarity | Rightward
(metA)- maLF I mal E
mal K
&16-
&17
polarity__. lam B .(uvrA) =
=
=
FIG. 1. Diagram of the malB region, adapted from Hofnung (19). The products of malE, malF, and malK are apparently involved in the transport of maltose (20). The product of malE is the maltose-binding protein (22) and the product of lamB is the receptor for the bacteriophage lambda (31).
207
here and in a subsequent publication. The evidence for an independent receptor that mediates taxis toward maltose has been summarized previously (4). That receptor detects no other sugar tested besides maltose. The receptor is highly inducible; i.e., bacteria grown on glycerol or D-galactose are attracted to maltose only slightly or not at all, but when grown in the presence of maltose they are attracted strongly. A mutant in malQ and one in malK were shown to be attracted normally to maltose, whereas a mutant that had the phenotype of a malT mutant showed no taxis toward the sugar. Thus, it can be suggested that at least one component necessary for maltose chemoreception is under the control of the malT product, which would account for the observed inducibility. MATERIALS AND METHODS Bacteria. All the bacterial strains used were obtained from M. Hofnung and M. Schwartz at the Institut Pasteur, Paris. Most are direct derivatives of E. coli K-12 strain HfrG6, which is His- (20). The lamB mutants are derived from pop 1048, a His+, metB, lacZam derivative HfrG6 (19). A motile derivative of each strain was selected from a swarm on a tryptone semisolid plate (7), and if that derivative was poorly motile when grown on minimal media a second selection was carried out on a minimal medium-galactose semisolid plate (16). Medium. The growth medium, containing mineral salts (Hi medium), glycerol (50 mM), and maltose (5 mM), has been described previously (2). Chemicals. Maltose (E. Merck, Darmstadt) was purified by descending chromatography on Whatman no. 54 paper with isopropanol-water (4:1) for 24 h at room temperature (36). This system clearly separates maltose from glucose and an unknown slower-moving component. D-Galactose (Sigma Chemical Co.) was "substantially glucose free." [l4C]maltose (Amersham, uniformly labeled) at 7 or 10 mCi/mmol was used for binding and uptake assays. The material was not purified further, but dilution with unlabeled purified maltose indicated that, within the error of the binding assay, the radioactivity was present solely as maltose. Glass-distilled water was used in making all solutions. Chemotaxis assay. The procedures of growth, preparation, and assay of the bacteria were essentially those described by Adler (2). Those procedures, along with the specific conditions used in this study, can be summarized as follows: Bacteria were grown at 35 C and washed at room temperature. A capillary (1-Al disposable micropipette) containing a solution of attractant was placed into a 0.3-ml suspension of bacteria (106 to 3 x 106 cells per ml) on a glass plate. The chemotaxis medium (10 mM potassium phosphate [pH 71 and 0.1 mM ethylenediaminetetraacetate) allows motility and chemotaxis but not growth; the bacteria rely on their endogenous energy source.
208
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HAZELBAUER
After incubation at 32 C for 45 min, the capillary was removed and the number of bacteria inside was determined by plating the contents (see Fig. 2 for examples). Under these conditions the relation between number of motile bacteria in the suspension and accumulation in the capillary is linear (2). The number of bacteria accumulating in a capillary containing only buffer was considered to be indicative of the number of motile bacteria in the suspension. That value was used to normalize the responses of different strains, as well as being subtracted as a "blank" from the value for accumulation in response to the presence of an attractant. All points in an assay were done at least in duplicate and the average value was plotted. (Examples of tactic response to maltose are shown in Fig. 2.) Maltose uptake assay. The initial rate of uptake of "4C-labeled maltose was determined by filtering 0.5-ml samples of a suspension of three-times-washed cells (5 x 107 cells per ml) on 0.45-Mm pore cellulose filters (Schleicher and Schiill, Dassel). A 3.5-ml volume of cells was incubated for 20 min at 30 C, [I4C ]maltose was added as a concentrated (100 times) solution, and samples were taken every 5 s for 30 s. The use of automatic pipettes and a sampling manifold (Millipore Corp.) made rapid sampling and immediate washing with 5 ml of chemotaxis medium possible. All solutions were at 30 C. The experiments were also done in the presence of 10 mM glycerol added 5 min before the addition of maltose. Filters were dried, and the retained radioactivity was determined by liquid scintillation counting. Values for nonspecific retention of maltose by the cells and filter were determined by using cells that had been first treated with 20% formaldehyde. Those values were subtracted from experimental values; they were less than 10% of the radioactivity retained by the wildtype cells 20 s after maltose addition. There is no known nonmetabolizable analogue for maltose, so that maltose transport can only be tested with the metabolizable sugar. Thus, the radioactivity accumulated in the cell includes nonmetabolized "soluble" maltose and metabolic products of the sugar, but does not include 14C that has been lost as 14CO2 during metabolism. In general, accumulation of radioactivity by wild-type strains was linear for the first 20 s (see Fig. 3), and then reached a plateau by 90 s. Within the limit of the scatter resulting from low accumulation of radioactivity, mutant uptake was linear over the 30-s sampling time. At 20 s more than 80% of the accumulated radioactivity could be removed from wild-type cells by a wash with chemotaxis medium containing 100 uM unlabeled maltose. At this time, less than 5% of the radioactivity had been lost from the incubation solution. The accumulated exchangeable maltose represents about a 700fold concentration at 20 s. Initial rates in Table 2 were determined from the linear portion of the uptake curves; 107 cells was equivalent to about 25 ag of cell wet weight. Osmotic shock procedure. Shock fluid was prepared from cells grown to late log phase (about 109 cells per ml) in 1.5 liters of minimal glycerol medium (containing 5 mM maltose and 0.05 mg of thiamine
per ml, as well as 0.1 mg of histidine or methionine per ml as required). The cells were washed and osmotically shocked by the procedure of Heppel (18). This involves transfer from a tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate-20% sucrose solution to ice-cold 0.5 mM MgCl2 and removal of the cells by centrifugation. The supernatant was made 10 mM in tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.3) and 2 mM in MgCl2, concentrated to about 2 ml by ultrafiltration through an Amicon UM-10 membrane, and then dialyzed against a solution of 10 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.3) and 2 mM MgCl2 (buffer A) at 4 C. Part of this solution was first fractionated by ammonium sulfate precipitation at 65% and then 95% saturation. Those fractions were dissolved in and dialyzed against buffer A. Maltose-binding assay. Binding of [14C]maltose to each of the three shock fluid fractions was determined by equilibrium dialysis as follows. Samples (0.1 ml) were placed in ethylenediaminetetraacetatewashed dialysis tubing, and dialyzed against 5 ml of [I4C]maltose in buffer A at concentrations of 0.1 to 100 MM. The protein concentration in the fractions was 1 to 4 mg/ml, and the concentration of maltose-binding sites was usually 5 to 20 MM. The dialysis was carried out at room temperature (23 C) for 6 to 8 h. The halftime of ['4C]maltose entry was on the order of 30 min under these conditions, and no loss of bound counts was seen at times up to 18 h. Occasional determination of protein in the sac before and after dialysis showed no significant changes. The concentration of free maltose was determined from the radioactivity present in a sample of maltose solution at equilibrium, and that of bound maltose was determined by the difference between the radioactivity in a sample from the sac and from the solution. Those values were plotted as a simple binding curve, an inverse plot, and as a Scatchard plot, and an estimate was made of the total concentration of maltose-binding sites. Over 90% of the total binding sites were in the 65 to 95% ammonium sulfate fraction. The sum of the sites in the two precipitated fractions was usually a bit larger than the number of sites determined for the unfractionated shock fluid. The presence of shock fluid from a mutant lacking binding activity did not inhibit the binding of an active shock fluid. At site concentrations of approximately 20 MM, none of the three plots was simple, and the latter two could be fitted with two straight lines from which could be determined two apparent dissociation constants of about 2 and 20 MuM (see Fig. 4). These observations are in accord with the characteristic binding of maltose to the pure maltose-binding protein determined by Kellermann and Szmelcman (22). Other assays. Protein was determined by the method of Lowry (24), with egg white lysozyme as a standard. The concentration of the purified maltose was determined by the method of Park and Johnson (30).
RESULTS Strains, isolated and characterized as carrying mutations in the genes of the maltose
VOL. 122, 1975
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by M. Hofnung and M. Schwartz, were tested for their taxis toward maltose. All strains were also tested for chemotaxis toward at at least one amino acid, usually L-aspartate, and one sugar, usually D-galactose. A few strains defective in maltose taxis were, in addition, examined for taxis toward compounds recognized by receptors other than the aspartate and the galactose receptor. The only significant difference observed between the taxis behavior of a wild-type parent strain and a mutant strain was taxis toward maltose. Maltose A region. It was shown previously that a malQ mutant is attracted normally to maltose, and a mutant phenotypically identified as defective in the control gene malT is not attracted at all (4). The mutation malP15 allows normal maltose taxis and the mutation malTl 73, which is known to map in malT, eliminates such taxis. Thus, the observation that the metabolism of maltose is not required for maltose taxis can include the functioning of maltodextrin phosphorylase as well as amylomaltase, and the control of the production of at least part of the maltose receptor by the malT product is confirmed. operons
Maltose B region. Table 1 includes a sumof maltose taxis behavior by mutants defective in genes of the malB region. Representative plots of normal and defective maltose taxis are shown in Fig. 2. To compare the response of various strains, taxis was quantitated by measuring the area under the response curve (after subtracting blank values) and normalizing to the number of motile bacteria present in the assay as determined by accumulation in a capillary containing only buffer. The derived value is expressed as a percentage of the wild-type value. The relation between the responses is not altered significantly if accumulation of bacteria at the peak is used instead of the area value or if normalization is accomplished by use of the magnitude of the response to an attractant other than maltose. The usual range of variation in these assays is approximately 20%. Most strains were also assayed for release of maltose-binding activity into their shock fluid (Table 1) and for initial rates of uptake of 1 and 10 AM maltose (Fig. 3 and Table 2). The efficiency of the shock procedure appears to be about equivalent for all strains tested as judged
mary
TABLE 1. Maltose taxis and maltose-binding activity Maltose taxis
Strain
Maltose mutation
Mutation characteristics
W
Wild-tepone response G6 pop 1048 pop 1744 pop 1748 pop 1749 pop 1750 pop 1752 pop 1753 pop 1754 pop 1755 pop 1756 pop 1757 pop 1758 pop 1759 pop 1760 pop 1761 pop 1729 pop 1730
pop 1051 pop 1053 pop 1054
None None malF2 malF6 malF7 malF8 malElO malE1l malE12 malE13 malE14 malE15
malE16 malE17 malKl malK3 malBA16 malBA17
lamB201 lamB203 lamB204
Amber Amber Amber Amber
Amber, polar Polar
Amber, polar Amber, nonpolar Polar deletion Deletion in malK extends into lam Amber Amber Amber
100 100 65 75 71 66 7 5 6 102 0 22 56
3 82 76 10 0 39 60 26
Peak concn (mM maltose)
1 1
Shock Fluid contentsa Maltosesites binding (nmolUmg of
protein) 3.0 3.0
Total protein (mg/g of wet cells)
2.8 3.4
1 0.5 1 0.02 0.02 1 5 10 10
+
+
0.2 1 0.3 1 0.3 1 1 0.3
Binder activity f
2.5
3.3
+
Vol. 122, No. 1
Printed in U.S.A.
Maltose Chemoreceptor of Escherichia coli GERALD L. HAZELBAUER The Wallenberg Laboratory, University of Uppsala, Uppsala, Sweden Received for publication 15 November 1974
Strains carrying mutations in the maltose system of Escherichia coli were assayed for maltose taxis, maltose uptake at 1 and 10 qM maltose, and maltose-binding activity released by osmotic shock. An earlier conclusion that the metabolism of maltose is not necessary for chemoreception is extended to include the functioning of maltodextrin phosphorylase, the product of malP, and the genetic control of the maltose receptor by the product of malT is confirmed. Mutants in malF and malK are defective in maltose transport at low concentrations as well as high concentrations, as previously shown, but are essentially normal in maltose taxis. The product of malE has been previously shown to be the maltose-binding protein and was implicated in maltose transport. Most malE mutants are defective in maltose taxis, and all those tested are defective in maltose transport at low concentrations. Thus, as previously suggested, the maltosebinding protein probably serves as the recognition component of the maltose receptor, as well as a component of the transport system. Some malE mutants release maltose-binding activity and are tactic toward maltose, although defective in maltose transport, implying that the binding protein has separate sites for interaction with the chemotaxis and transport systems. Some mutations in lamB, whose product is the receptor for the bacteriophage lamba, cause defects in maltose taxis, indicating some involvement of that product in maltose reception.
The phenomenon of chemotaxis by Escherichia coli appears to result from the effect that a temporal gradient of an active compound has on the direction of rotation of bacterial flagella (23). Counter-clockwise rotation of flagella produces smooth swimming, and clockwise rotation produces a "tumble" (8, 9, 35) which results in a change of direction when swimming is resumed (10). An increase in attractant concentration shifts the balance between these two modes to smooth swimming (10, 13, 25), whereas a drastic decrease shifts it to tumbling (25). The effect of an increase or decrease in repellent concentration is the opposite from that of an attractant (23, 37, 38). Thus, bacteria tend to move up a spatial gradient of attractant and down one of repellent. Changes in concentration of chemotactically active compounds are detected by specific "chemoreceptors." Adler demonstrated that the functioning of these chemoreceptors does not require metabolism or general transport of the compound, but only requires the recognition of the molecule, presumably by a specific binding (1). In the simplest case, a given chemoreceptor includes all those structures involved in the
detection of one class of compounds and not in the detection of other classes. (There is genetic evidence that some chemoreceptors "overlap" [29; J. Adler, personal communication].) Receptors are linked to a "common pathway" through which recognition is translated into an effect on the flagella (1). Mutants are available that are defective in a particular chemoreceptor (4, 17, 26, 28, 29, 38). Such mutants, defective in the galactose receptor, made possible the identification of the galactose-binding protein (6) as the recognition component of that receptor (16). Boos has shown that this protein is a part of the 3-methylgalactoside transport system (11), which transports low concentrations of galactose into the cell (21, 33). The galactose-binding protein is released from the envelope of E. coli by osmotic shock, i.e., it is a periplasmic protein (18). A search for other shock-released binding proteins that might serve as chemoreceptor recognition components produced a ribose-binding activity and a maltose-binding activity (16). Later, a ribosebinding protein which probably functions in a ribose receptor was purified from Salmonella typhimurium (5). The maltose-binding activity
206
VOL. 122, 1975
MALTOSE CHEMORECEPTOR OF E. COLI
was purified as a maltose-binding protein and shown to be the product of the gene malE by Kellermann and Szmelcman (22). Recognition components for the glucose and mannose receptors have been identified as enzymes II of the phosphotransferase transport system by the characterization of the chemotactic behavior of enzyme II mutants and preliminary results indicate that the enzymes II for D-fructose and D-mannitol serve as the receptors for those two sugars, respectively (3). Genetic studies indicate that the galactose receptor has a second component in addition to the binding protein (28). These biochemical and genetic identifications constitute the information presently available about bacterial chemoreceptors. This paper is a beginning of the characterization of the maltose receptor of E. coli. Here the involvement in taxis toward maltose of the gene products of the known maltose operons is determined. Mutations that specifically affect the metabolism of maltose (thus excluding mutations in the catabolite repression system and the phosphotransferase system) are grouped in the two regions on the genetic map of E. coli (34). The malA region at 65 min contains malT, which codes for the positive regulatory molecule controlling all three maltose operons, and an operon consisting of malP, which codes for maltodextrin phosphorylase, and malQ, which codes for amylomaltase (34). The malB region at 79 min (Fig. 1) contains two divergent operons which have adjoining, and perhaps overlapping, promotor regions (19, 20). Mutants in malE, malF, or malK are defective in maltose transport and thus are unable to grow on maltose as a carbon source. The maltose-binding protein is the product of malE (22). The product of lamB is a protein that serves as the outer membrane receptor for the bacteriophage lambda (31). This protein is apparently also involved in uptake of low concentrations of maltose (M. Hofnung, personal communication). The involvement of the lambda receptor in maltose chemoreception will be discussed Leftward poLarity | Rightward
(metA)- maLF I mal E
mal K
&16-
&17
polarity__. lam B .(uvrA) =
=
=
FIG. 1. Diagram of the malB region, adapted from Hofnung (19). The products of malE, malF, and malK are apparently involved in the transport of maltose (20). The product of malE is the maltose-binding protein (22) and the product of lamB is the receptor for the bacteriophage lambda (31).
207
here and in a subsequent publication. The evidence for an independent receptor that mediates taxis toward maltose has been summarized previously (4). That receptor detects no other sugar tested besides maltose. The receptor is highly inducible; i.e., bacteria grown on glycerol or D-galactose are attracted to maltose only slightly or not at all, but when grown in the presence of maltose they are attracted strongly. A mutant in malQ and one in malK were shown to be attracted normally to maltose, whereas a mutant that had the phenotype of a malT mutant showed no taxis toward the sugar. Thus, it can be suggested that at least one component necessary for maltose chemoreception is under the control of the malT product, which would account for the observed inducibility. MATERIALS AND METHODS Bacteria. All the bacterial strains used were obtained from M. Hofnung and M. Schwartz at the Institut Pasteur, Paris. Most are direct derivatives of E. coli K-12 strain HfrG6, which is His- (20). The lamB mutants are derived from pop 1048, a His+, metB, lacZam derivative HfrG6 (19). A motile derivative of each strain was selected from a swarm on a tryptone semisolid plate (7), and if that derivative was poorly motile when grown on minimal media a second selection was carried out on a minimal medium-galactose semisolid plate (16). Medium. The growth medium, containing mineral salts (Hi medium), glycerol (50 mM), and maltose (5 mM), has been described previously (2). Chemicals. Maltose (E. Merck, Darmstadt) was purified by descending chromatography on Whatman no. 54 paper with isopropanol-water (4:1) for 24 h at room temperature (36). This system clearly separates maltose from glucose and an unknown slower-moving component. D-Galactose (Sigma Chemical Co.) was "substantially glucose free." [l4C]maltose (Amersham, uniformly labeled) at 7 or 10 mCi/mmol was used for binding and uptake assays. The material was not purified further, but dilution with unlabeled purified maltose indicated that, within the error of the binding assay, the radioactivity was present solely as maltose. Glass-distilled water was used in making all solutions. Chemotaxis assay. The procedures of growth, preparation, and assay of the bacteria were essentially those described by Adler (2). Those procedures, along with the specific conditions used in this study, can be summarized as follows: Bacteria were grown at 35 C and washed at room temperature. A capillary (1-Al disposable micropipette) containing a solution of attractant was placed into a 0.3-ml suspension of bacteria (106 to 3 x 106 cells per ml) on a glass plate. The chemotaxis medium (10 mM potassium phosphate [pH 71 and 0.1 mM ethylenediaminetetraacetate) allows motility and chemotaxis but not growth; the bacteria rely on their endogenous energy source.
208
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HAZELBAUER
After incubation at 32 C for 45 min, the capillary was removed and the number of bacteria inside was determined by plating the contents (see Fig. 2 for examples). Under these conditions the relation between number of motile bacteria in the suspension and accumulation in the capillary is linear (2). The number of bacteria accumulating in a capillary containing only buffer was considered to be indicative of the number of motile bacteria in the suspension. That value was used to normalize the responses of different strains, as well as being subtracted as a "blank" from the value for accumulation in response to the presence of an attractant. All points in an assay were done at least in duplicate and the average value was plotted. (Examples of tactic response to maltose are shown in Fig. 2.) Maltose uptake assay. The initial rate of uptake of "4C-labeled maltose was determined by filtering 0.5-ml samples of a suspension of three-times-washed cells (5 x 107 cells per ml) on 0.45-Mm pore cellulose filters (Schleicher and Schiill, Dassel). A 3.5-ml volume of cells was incubated for 20 min at 30 C, [I4C ]maltose was added as a concentrated (100 times) solution, and samples were taken every 5 s for 30 s. The use of automatic pipettes and a sampling manifold (Millipore Corp.) made rapid sampling and immediate washing with 5 ml of chemotaxis medium possible. All solutions were at 30 C. The experiments were also done in the presence of 10 mM glycerol added 5 min before the addition of maltose. Filters were dried, and the retained radioactivity was determined by liquid scintillation counting. Values for nonspecific retention of maltose by the cells and filter were determined by using cells that had been first treated with 20% formaldehyde. Those values were subtracted from experimental values; they were less than 10% of the radioactivity retained by the wildtype cells 20 s after maltose addition. There is no known nonmetabolizable analogue for maltose, so that maltose transport can only be tested with the metabolizable sugar. Thus, the radioactivity accumulated in the cell includes nonmetabolized "soluble" maltose and metabolic products of the sugar, but does not include 14C that has been lost as 14CO2 during metabolism. In general, accumulation of radioactivity by wild-type strains was linear for the first 20 s (see Fig. 3), and then reached a plateau by 90 s. Within the limit of the scatter resulting from low accumulation of radioactivity, mutant uptake was linear over the 30-s sampling time. At 20 s more than 80% of the accumulated radioactivity could be removed from wild-type cells by a wash with chemotaxis medium containing 100 uM unlabeled maltose. At this time, less than 5% of the radioactivity had been lost from the incubation solution. The accumulated exchangeable maltose represents about a 700fold concentration at 20 s. Initial rates in Table 2 were determined from the linear portion of the uptake curves; 107 cells was equivalent to about 25 ag of cell wet weight. Osmotic shock procedure. Shock fluid was prepared from cells grown to late log phase (about 109 cells per ml) in 1.5 liters of minimal glycerol medium (containing 5 mM maltose and 0.05 mg of thiamine
per ml, as well as 0.1 mg of histidine or methionine per ml as required). The cells were washed and osmotically shocked by the procedure of Heppel (18). This involves transfer from a tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetate-20% sucrose solution to ice-cold 0.5 mM MgCl2 and removal of the cells by centrifugation. The supernatant was made 10 mM in tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.3) and 2 mM in MgCl2, concentrated to about 2 ml by ultrafiltration through an Amicon UM-10 membrane, and then dialyzed against a solution of 10 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.3) and 2 mM MgCl2 (buffer A) at 4 C. Part of this solution was first fractionated by ammonium sulfate precipitation at 65% and then 95% saturation. Those fractions were dissolved in and dialyzed against buffer A. Maltose-binding assay. Binding of [14C]maltose to each of the three shock fluid fractions was determined by equilibrium dialysis as follows. Samples (0.1 ml) were placed in ethylenediaminetetraacetatewashed dialysis tubing, and dialyzed against 5 ml of [I4C]maltose in buffer A at concentrations of 0.1 to 100 MM. The protein concentration in the fractions was 1 to 4 mg/ml, and the concentration of maltose-binding sites was usually 5 to 20 MM. The dialysis was carried out at room temperature (23 C) for 6 to 8 h. The halftime of ['4C]maltose entry was on the order of 30 min under these conditions, and no loss of bound counts was seen at times up to 18 h. Occasional determination of protein in the sac before and after dialysis showed no significant changes. The concentration of free maltose was determined from the radioactivity present in a sample of maltose solution at equilibrium, and that of bound maltose was determined by the difference between the radioactivity in a sample from the sac and from the solution. Those values were plotted as a simple binding curve, an inverse plot, and as a Scatchard plot, and an estimate was made of the total concentration of maltose-binding sites. Over 90% of the total binding sites were in the 65 to 95% ammonium sulfate fraction. The sum of the sites in the two precipitated fractions was usually a bit larger than the number of sites determined for the unfractionated shock fluid. The presence of shock fluid from a mutant lacking binding activity did not inhibit the binding of an active shock fluid. At site concentrations of approximately 20 MM, none of the three plots was simple, and the latter two could be fitted with two straight lines from which could be determined two apparent dissociation constants of about 2 and 20 MuM (see Fig. 4). These observations are in accord with the characteristic binding of maltose to the pure maltose-binding protein determined by Kellermann and Szmelcman (22). Other assays. Protein was determined by the method of Lowry (24), with egg white lysozyme as a standard. The concentration of the purified maltose was determined by the method of Park and Johnson (30).
RESULTS Strains, isolated and characterized as carrying mutations in the genes of the maltose
VOL. 122, 1975
209
MALTOSE CHEMORECEPTOR OF E. COLI
by M. Hofnung and M. Schwartz, were tested for their taxis toward maltose. All strains were also tested for chemotaxis toward at at least one amino acid, usually L-aspartate, and one sugar, usually D-galactose. A few strains defective in maltose taxis were, in addition, examined for taxis toward compounds recognized by receptors other than the aspartate and the galactose receptor. The only significant difference observed between the taxis behavior of a wild-type parent strain and a mutant strain was taxis toward maltose. Maltose A region. It was shown previously that a malQ mutant is attracted normally to maltose, and a mutant phenotypically identified as defective in the control gene malT is not attracted at all (4). The mutation malP15 allows normal maltose taxis and the mutation malTl 73, which is known to map in malT, eliminates such taxis. Thus, the observation that the metabolism of maltose is not required for maltose taxis can include the functioning of maltodextrin phosphorylase as well as amylomaltase, and the control of the production of at least part of the maltose receptor by the malT product is confirmed. operons
Maltose B region. Table 1 includes a sumof maltose taxis behavior by mutants defective in genes of the malB region. Representative plots of normal and defective maltose taxis are shown in Fig. 2. To compare the response of various strains, taxis was quantitated by measuring the area under the response curve (after subtracting blank values) and normalizing to the number of motile bacteria present in the assay as determined by accumulation in a capillary containing only buffer. The derived value is expressed as a percentage of the wild-type value. The relation between the responses is not altered significantly if accumulation of bacteria at the peak is used instead of the area value or if normalization is accomplished by use of the magnitude of the response to an attractant other than maltose. The usual range of variation in these assays is approximately 20%. Most strains were also assayed for release of maltose-binding activity into their shock fluid (Table 1) and for initial rates of uptake of 1 and 10 AM maltose (Fig. 3 and Table 2). The efficiency of the shock procedure appears to be about equivalent for all strains tested as judged
mary
TABLE 1. Maltose taxis and maltose-binding activity Maltose taxis
Strain
Maltose mutation
Mutation characteristics
W
Wild-tepone response G6 pop 1048 pop 1744 pop 1748 pop 1749 pop 1750 pop 1752 pop 1753 pop 1754 pop 1755 pop 1756 pop 1757 pop 1758 pop 1759 pop 1760 pop 1761 pop 1729 pop 1730
pop 1051 pop 1053 pop 1054
None None malF2 malF6 malF7 malF8 malElO malE1l malE12 malE13 malE14 malE15
malE16 malE17 malKl malK3 malBA16 malBA17
lamB201 lamB203 lamB204
Amber Amber Amber Amber
Amber, polar Polar
Amber, polar Amber, nonpolar Polar deletion Deletion in malK extends into lam Amber Amber Amber
100 100 65 75 71 66 7 5 6 102 0 22 56
3 82 76 10 0 39 60 26
Peak concn (mM maltose)
1 1
Shock Fluid contentsa Maltosesites binding (nmolUmg of
protein) 3.0 3.0
Total protein (mg/g of wet cells)
2.8 3.4
1 0.5 1 0.02 0.02 1 5 10 10
+
+
0.2 1 0.3 1 0.3 1 1 0.3
Binder activity f
2.5
3.3
+
